The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants.

In 2000, the thematic network ENTRANSFOOD was launched to assess four different topics that are all related to the testing or assessment of food containing or produced from genetically modified organisms (GMOs). Each of the topics was linked to a European Commission (EC)-funded large shared cost action (see http://www.entransfood.com). Since the exchange of genetic information through horizontal (lateral) gene transfer (HGT) might play a more important role, in quantity and quality, than hitherto imagined, a working group dealing with HGT in the context of food and feed safety was established. This working group was linked to the GMOBILITY project (GMOBILITY, 2003) and the results of the deliberations are laid down in this review paper. HGT is reviewed in relation to the potential risks of consuming food or feed derived from transgenic crops. First, the mechanisms for obtaining transgenic crops are described. Next, HGT mechanisms and its possible evolutionary role are described. The use of marker genes is presented in detail as a special case for genes that may pose a risk. Furthermore, the exposure to GMOs and in particular to genetically modified (GM) deoxyribonucleic acid (DNA) is discussed as part of the total risk assessment. The review finishes off with a number of conclusions related to GM food and feed safety. The aim of this paper is to provide a comprehensive overview to assist risk assessors as well as regulators and the general public in understanding the safety issues related to these mechanisms.

[Outpatient rehabilitation of alcohol dependent patients in a day clinic: initial results of a comparative follow-up study of day clinic and inpatient treatment]. / Ambulante Rehabilitation alkoholabhängiger Patienten in einer Tagesklinik: Erste Ergebnisse einer vergleichenden katamnestischen Untersuchung von tagesklinischer und stationärer Behandlung.

This article describes the development of an outpatient concept for the day-clinic withdrawal treatment of alcohol dependent persons in a Hamburg institution for addiction aid. It reports the initial, selected results of a comparative follow-up study of patients discharged during the years 1998-2000, who either receive ambulatory treatment in the day clinic (n = 270) or inpatient treatment in the institution's special clinic (n = 462). Assessable questionnaires are available from 131 outpatients of the day-clinic treatment and 173 patients of the inpatient treatment form. The response rate - with reference to the group of those who were reachable - was 57.2 % for patients of the day clinic and 53.2 % for patients of the special clinic. The results of the study arrive at the conclusion that both treatment forms can be seen as thoroughly comparable with regard to primary outcome measurements (for example reduction of psychological stress, abstinence rates, reintegration into occupational life). Rehabilitants treated in the inpatient setting more frequently report that they had already contacted centres for further treatment and self-help groups during the rehabilitation phase, which however doesn't lead to a change of participation behaviour following the rehabilitation phase. This serves to confirm the assumption that an additional offer of a day-clinic service in the area of addiction rehabilitation provides a further, effective treatment concept that sensibly supplements the otherwise inpatient-oriented treatment landscape. The results indicate the quality of the work performed in the day clinic studied (as well as in the inpatient clinic) and should encourage the funding agencies and employees of other day clinic institutions in the field of addiction rehabilitation to participate in evaluation and quality assurance measures, thus continuing to bridge the gap between the (theoretical) state of knowledge concerning outpatient rehabilitation and the degree to which it can be successfully realized.

Genome sequence of bovine herpesvirus 4, a bovine Rhadinovirus, and identification of an origin of DNA replication.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus of cattle. The complete long unique coding region (LUR) of BoHV-4 strain 66-p-347 was determined by a shotgun approach. Together with the previously published noncoding terminal repeats, the entire genome sequence of BoHV-4 is now available. The LUR consists of 108,873 bp with an overall G+C content of 41.4%. At least 79 open reading frames (ORFs) are present in this coding region, 17 of them unique to BoHV-4. In contrast to herpesvirus saimiri and human herpesvirus 8, BoHV-4 has a reduced set of ORFs homologous to cellular genes. Gene arrangement as well as phylogenetic analysis confirmed that BoHV-4 is a member of the genus Rhadinovirus. In addition, an origin of replication (ori) in the genome of BoHV-4 was identified by DpnI assays. A minimum of 1.69 kbp located between ORFs 69 and 71 was sufficient to act as a cis signal for replication.

[Thought protocols before and after a psychological headache--treatment with imagery]. / Gedankenprotokolle vor und nach einer psychologischen Schmerztherapie mit Imaginationsübungen.

Cognitive changes accompanying a headache treatment with imagery strategies were investigated. 15 female and 6 male headache sufferers with migraine (6), tension-type headache (7) or both (8) wrote down their thoughts during 1-5 headache episodes, kept a headache diary and filled in several pain questionnaires before and after treatment. After treatment there were significantly more positive and less negative thoughts. Pain frequency, duration, medication intake intensity, catastrophizing and helplessness were reduced, self-efficacy was enhanced. Changes of thought protocol variables were correlated with treatment effects. The role of cognitive changes in the psychological treatment of chronic headache is discussed.

A sensitive and specific PCR/Southern blot assay for detection of bovine herpesvirus 4 in calves infected experimentally.

A PCR/Southern blot assay for detection of bovine herpesvirus 4 (BHV-4) in the background of bovine cellular DNA was developed. A BHV-4 specific sequence within the gene coding for the glycoprotein B (gB) was selected for primer sequences to guarantee the specificity of the assay. With a detection limit of six molecules BHV-4 DNA in the background of 1 microg of cellular DNA (equals about 150,000 bovine cells) this PCR/Southern blot assay represents a highly sensitive method for detection of BHV-4 DNA. At low concentrations of BHV-4 genomes, this assay also allows to estimate the copy number of BHV-4: a distinction between fewer than 6, 6-59 and more than 60 BHV-4 genomes/100 microl DNA suspension was possible. Tissue and blood samples of two calves, infected experimentally with BHV-4 were examined for the prevalence of BHV-4 DNA 130 days post infection. Ten days before taking samples, one of the calves was immuno-suppressed with dexamethasone. In both calves, BHV-4 DNA was detected in the leucocyte fraction of the blood, and beyond that in lower quantities in the spleen and the kidney of the immuno-suppressed calf. It is assumed that a latent BHV-4 infection was activated after application of dexamethasone and that the leucocyte fraction of the blood represents one site of latency of BHV-4 in cattle.

Genetic analysis of the bovine herpesvirus type 4 gene locus for the putative terminase.

The complete DNA sequence of the 10-45 kbp HindIII B fragment of bovine herpesvirus type 4 (BoHV-4) was determined. This fragment contains nine complete and two incomplete open reading frames (ORFs), all of which are homologous to herpesvirus saimiri (HVS), Kaposi's Sarcoma-associated herpesvirus (HHV-8) and Epstein-Barr virus (EBV). Particularly, the arrangement of the gene for the terminase-related protein with the two coding exons 29a/29b is conserved among all herpesviruses sequenced to date. The intron carries the ORFs 30 to 33 in the opposite direction. Analysis by reverse transcription and polymerase chain reaction (PCR) of the transcript across the proposed splice junction of the ORF 29a/29b and subsequent sequence determination of the amplified product revealed the precise structure of the splice junction. Furthermore, the phylogenetic analysis of the 29a/29b protein and its counterparts in other herpesviruses revealed that BoHV-4 clustered in the genus Rhadinovirus of the subfamily Gammaherpesvirinae.

Detection of new DNA polymerase genes of known and potentially novel herpesviruses by PCR with degenerate and deoxyinosine-substituted primers.

A consensus primer PCR approach was used to (i) investigate the presence of herpesviruses in wild and zoo equids (zebra, wild ass, tapir) and to (ii) study the genetic relationship of the herpesvirus of pigeons (columbid herpesvirus 1) to other herpesvirus species. The PCR assay, based on degenerate primers targeting highly conserved regions of the DNA polymerase gene of herpesviruses, was modified by using a mixture of degenerate and deoxyinosine-substituted primers. The applicability of the modification was validated by amplification of published DNA polymerase genes of 16 herpesvirus species and of the previously uncharacterized DNA polymerase genes of equine herpesvirus 3 (EHV-3) and equine herpesvirus 5 (EHV-5). The modified assay was then used for partial amplification of the polymerase of columbid herpesvirus 1 which is presently classified as a beta-herpesvirus based on biological criteria. Sequence analysis of amplicons obtained from four different viral strains revealed a close relationship of columbid herpesvirus 1 to members of the subfamily Alphaherpesvirinae, especially to Marek's disease herpesvirus. This was confirmed by characterization of additional 1.6kb of the columbid herpesvirus 1 polymerase. Consensus PCR analysis of blood samples from zebras, a wild ass and a tapir revealed amplicons showing high percentages ( > 50%) of sequence identity to DNA polymerases of gamma-herpesviruses. In particular, the zebra and the wild ass sequence were closely related to each other and to the polymerases of the equine gamma-herpesviruses EHV-2 and EHV-5 with sequence identities of > 80%. This is a first indication that novel gamma-herpesviruses are present in wild and zoo equids.

Structure and function of the prDNA and the genomic termini of the gamma2-herpesvirus bovine herpesvirus type 4.

The linear virion DNA of bovine herpesvirus type 4 (BHV-4) is flanked by tandem repeats designated polyrepetitive DNA (prDNA). To investigate the structure and functional role of the prDNA for cleavage/packaging of progeny viral DNA, the complete nucleotide sequence (2267 bp) of a cloned prDNA unit of BHV-4 was determined. Moreover, the terminal fragments of the genome and the junctions between prDNA and the central unique DNA were analysed. In order to characterize the function of the prDNA of BHV-4, a transient packaging assay was developed. The prDNA has a G+C content of 71.1%. Its structure is composed of numerous internal repeats and every unit contains the conserved sequence of the cleavage/packaging signal. A fragment of 443 bp comprising the cleavage/packaging signal was found to be sufficient for cleavage and encapsidation of replicated concatemeric viral DNA. These results suggest that prDNA is a functionally important region of the genome of BHV-4.

Transcription analysis of porcine circovirus (PCV).

This study focuses on gene expression of porcine circovirus (PCV) in order to identify viral genes and their corresponding mRNA transcripts. By northern blot analysis, the existence of three mRNAs could be demonstrated. Two mRNAs are encoded by the viral (-)-strand and one is encoded by the viral (+)-strand. The (+)-strand encoded mRNA transcript is 990 nucleotides (nt) long and corresponds to the open reading frame (ORF) 1, as shown by S1 mapping. The start point of this transcript is located at pos. 1238, as determined by primer extension analysis and rapid amplification of cDNA ends (RACE). The transcript is spliced as shown by direct reverse sequencing and RACE. It contains an untranslated "leader"-sequence 119 nt in size (pos. 1238 to 1120) which is joined to exon 2 of the ORF 1 transcript at pos. 737. The transcriptional regulatory elements have been identified functionally by CAT assays. They are located within a 258 base points (bp) fragment (pos. 1168 to 1425).

Identification of a protein essential for replication of porcine circovirus.

The largest open reading frame of porcine circovirus (ORF 4) encodes a protein of 312 amino acids. The predicted gene product of ORF 4 shows similarities to Rep proteins of other plant circoviruses and geminiviruses. Three motifs have been identified that are characteristic for proteins involved in rolling circle replication and the consensus sequence for a putative dNTP-binding box (GKS) has been found. In this paper, experimental evidence is presented which indicates that ORF 4 encodes the replication protein of porcine circovirus. After cloning of the ORF 4 gene product, it was supplied in trans in a transient replication assay. The ORF 4 gene product promoted the replication of plasmid pOP11, which carries the origin of DNA replication of porcine circovirus. Since pOP11 itself is unable to replicate in virus-free porcine kidney cells, the ORF 4 gene product must be essential for replication of porcine circovirus.

Mapping and characterization of the origin of DNA replication of porcine circovirus.

The origin of DNA replication of porcine circovirus (PCV) was mapped to a 111-bp fragment. On top of a hairpin, a nonanucleotide (TAGTATTAC) homologous to nonanucleotides of other viruses was identified. Mutation of this element abolishes replication. PCV may be related to a virus family characterized by single-stranded circular DNA genomes, rolling-circle replication, and homology of their rep proteins.

[Psychophysiologic and psychoneuroimmunologic studies in neurodermatitis. Overview and critical evaluation]. / Psychophysiologische und psychoneuroimmunologische Ergebnisse zur Neurodermitis. Ubersicht und kritische Bilanz.

Results of psychophysiological, psychoendocrinological, and psychoneuroimmunological research on the skin in patients with atopic dermatitis were evaluated. 11 investigations were selected and analysed with respect to both design and results. In 6 instances, healthy or ill control groups were included, rarely did the sample size exceed 30. With respect to physiology, blood pressure, heart rate and EDA were most commonly assessed; with respect to immunology, number of leucocytes and differential blood count and with respect to psychology, anxiety, neuroticism and stress perception. The results involving stress induction, itching induction and the relationship of personality and skin parameters were not consistent. The best established relationship is that between skin reactivity (flare, wheal size and pruritus) on the one hand and cognitive appraisal of stress-stimuli and the experimental situation on the other hand. Psychoendocrinological and even more psychoimmunological indicators of the stress of reaction-unlike psychophysiological indicators-were correlated with the skin response. Only half of the studies found an elevated physiological stress reaction in patients with atopic dermatitis.

The replication in vitro of the gammaherpesvirus bovine herpesvirus 4 is restricted by its DNA synthesis dependence on the S phase of the cell cycle.

Because several observations have suggested that replication of the gammaherpesvirus bovine herpesvirus 4 (BHV-4) is influenced by the physiological state of the host cell, a study was carried out to determine the relationship between BHV-4 infection and the cell cycle. The temporal expression of BHV-4 late (L) proteins in unsynchronized cell cultures was first investigated by flow cytometry. Interestingly, L protein expression occurred in a limited number of cells infected with a high multiplicity of infection, and a reciprocal correlation between the percentage of positive cells and the cell density at the time of infection was demonstrated. Moreover, the finding that a BHV-4 early-late protein was expressed in nearly all the cells suggested that a blockage in the viral replication cycle occurred in some infected cells at the stage of viral DNA synthesis or L protein expression. Because this blockage could be the consequence of the dependence of one or both of these events on the cell cycle, they were investigated after infection of synchronized cell cultures. The following findings were made. (i) Cell transition through the S phase quantitatively increased the rate of BHV-4 DNA replication. (ii) BHV-4 DNA synthesis could not be detected in cells arrested in G0. (iii) Synchronization of MDBK cells with Lovastatin before infection increased the percentage of cells expressing L proteins. (iv) In contrast, infection of cells arrested in G0 led to few positive cells. Taken together these results showed that BHV-4 DNA replication and consequently the expression of L proteins are dependent on the S phase of the cell cycle. This dependence could be of importance for several biological properties of BHV-4 infection in vitro and might have implications for the biology of the virus in vivo.

Glycoprotein B of bovine herpesvirus type 4: its phylogenetic relationship to gB equivalents of the herpesviruses.

In order to estimate the phylogenetic relationship of BHV-4 among the herpesviruses, we have cloned and sequenced its glycoprotein B (gB). The 2.6 kb open reading frame codes for a 874 amino acid long protein. The comparison of its deduced amino acid sequence with those of its counterparts in 19 distinct herpesviruses groups BHV-4 into the gamma-herpesvirinae. The calculation of an evolutionary tree emphasized that BHV-4 is more closely related to herpesvirus saimiri (HVS) than to Epstein-Barr virus (EBV). However, in contrast to EBV and HVS, the gB of BHV-4 contains a putative protease cleavage site and 20 potential N-glycosylation sites. The alignment of the amino acid sequences revealed that 10 cysteine and 7 proline residues, as well as the motifs SPF and GQLG, were completely conserved among the 20 investigated gBs.

Conserved domains of glycoprotein B (gB) of the monkey virus, simian agent 8, identified by comparison with herpesvirus gBs.

The herpesvirus simian agent 8 (SA8) gene which corresponds to the herpes simplex virus (HSV) gene encoding glycoprotein B (gB) was localized, cloned and sequenced. Comparison of its deduced amino acid sequence with those of its counterparts in 12 other distinct herpesvirus was used to evaluate their homology and phylogenetic relationship. The results emphasized that SA8 gB is more closely related to those of HSV-1 and -2, and bovine herpesvirus 2 than to the homologous proteins of other herpesviruses. Furthermore, the alignment showed several regions of domains conserved in the closely related sequences, including four conserved in all the herpesvirus gB sequences examined. The conservation of 10 cysteine residues and most of the proline residues, as well as several potential N-glycosylation sites, suggested that the secondary and tertiary structures of these gBs were similar.

Recombination of genomic terminus of bovine herpesvirus type 1 with cellular DNA.

Bovine herpesvirus 1 (BHV-1) has a linear DNA genome of about 135 kb which appears as two isomers, resulting from its short unique segment being present in the two possible orientations with respect to the large unique segment. BHV-1 also circularizes its DNA to form replicative molecules. Definition of the target sequences at the genomic termini involved in the recombination events during genomic replication and isomerization, as well as virus maturation, led to the discovery that 10% of the genome molecules have additional DNA sequences attached to the right-hand terminus, as shown by electron microscopy. Three such tails have been cloned molecularly; they differ in length and nucleotide sequence, and hybridization experiments demonstrate the cellular origin of two of the three tails. The evidence presented here is consistent with a proportion of the BHV-1 genomes recombining their DNA with cellular DNA during lytic infection.

Genomic termini of equine herpesvirus 1.

After cell infection with the equine herpesvirus 1 (EHV-1), the termini of the linear double-stranded DNA genome fuse to form circular forms. To investigate the mechanisms in the generation and cleavage of such replicative-form DNAs, the genomic termini, the fusion of termini from replicative-form molecules, and the junction between the short and long genome segments have been analyzed by restriction mapping, blot hybridizations, cloning, and sequencing. The data suggest that the genome ends are not redundant and that the genomic termini are fused in replicative intermediates via 3' single-base extensions at the termini of the unique long segment (UL) and terminal repeat (TR). Adjacent to the EHV-1 termini are AT and gamma sequence elements highly conserved among different herpesviruses. We propose that both of these sequence elements are important for the cleavage of EHV-1 replicative forms.

Non-C-G recognition sequences of DNA cytosine-5-methyltransferase from rat liver.

The eukaryotic DNA cytosine-5-methyltransferase (E.C.2.1.1.37) is known to methylate cytosine in DNA mainly, but not exclusively in C-G. In the present study the minor, non-C-G recognition sequences of a rat DNA methyltransferase were analyzed by Maxam-Gilbert sequencing of in vitro methylated SV40 DNA. The enzyme methylates C-A and C-T at a 50-fold lower initial rate than C-G. Methylation of C-C at the 5'C was not observed in the piece of DNA sequenced. The methylation of C-A is very low in the trinucleotides ACA and CAC, the other C-A containing trinucleotides in DNA are much better methylacceptors. C-T was found methylated predominantly in the sequences CCTAA, ACTAA, and ACTGT. A comparison of the activity with different substrates is in favour of the enzyme making its recognition in the major groove of the DNA.

Replication of single-stranded porcine circovirus DNA by DNA polymerases alpha and delta.

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64-66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39-57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase alpha and delta as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase alpha and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase alpha holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789-4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase alpha is blocked with the DNA polymerase alpha specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase delta can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.